Three new and exciting findings from this laboratory from the foundation for this proposal. 1) We demonstrated that Kupffer cells participate in the mechanism of elevated oxygen metabolism in hepatic parenchymal cells caused by acute ethanol treatment, 2) that inactivation of Kupffer cells prevents early injury due to ethanol in the Tsukamoto-French model, and 3) that hypoxia and free radicals are formed in vivo in response to ethanol in this model. Collectively, this new work has led us to hypothesize that early alcohol-induced liver damage is due to an oxygen- dependent reperfusion injury involving both hypoxia due to hypermetabolism and/or impaired microcirculation with subsequent O2- dependent free radical formation. We are eager to test this hypothesis using the clinical relevant Tsukamoto-French model of alcohol treatment exclusively employing a combination of specialized techniques (e.g. miniature O2 electrodes) unique to this laboratory. This will allow us to fill critical gaps in our knowledge which will lead to our important long-term goal -- the prevention of early alcohol-induced liver injury in the alcoholic. The first major goal will be to determine if hepatic nonparenchymal cells are involved in regulation of O2 uptake and if they can explain alcohol-induced hypoxia due to chronic ethanol treatment. Kupffer and endothelial cells will be selectively inactivated and the effect of chronic alcohol treatment and oxygen concentration on oxygen uptake will be assessed in the perfused liver at two week intervals for up to 4 months during development of early liver disease in Tsukamoto- French rats. We recently demonstrated that cultured Kupffer cells produce mediators which stimulate oxygen uptake in parenchymal cells, so we will determine which eicosanoids and/or selected cytokines produced by Kupffer cells are involved in an oxygen sensor mechanism which is stimulated by O2 and Tsukamoto-French ethanol treatment. The possible role of Kupffer cell Ca2+ channels in the O2 sensing mechanism will be assessed from depolarization-induced Ca2+ influx determined fluorometrically in cultured Kupffer cells. Our second major goal will be to determine if an oxygen-dependent reperfusion injury is a critical event in early alcohol-induced liver injury. The role of Kupffer cells in an oxygen-dependent reperfusion injury in a low-flow, reflow model of liver perfusion will be assessed. Subsequently, we will exploit temporal fluctuations in blood ethanol in the Tsukamoto-French model in vivo to dissect components of a reperfusion injury. When blood ethanol is high, we expect to detect hypoxia with miniature surface O2 electrodes as well as purine accumulation due to inefficient energetics predominantly in pericentral regions of the liver lobule. We predict that free radicals will be formed as blood ethanol declines and oxygen reenters the previously hypoxic tissue. Collectively, the new approach embodied in these experiments will link hypoxia and free radicals in the mechanism of alcohol-induced liver injury. By identifying the role of O2-dependent reperfusion injury and the role of nonparenchymal cells, a deeper understanding of mechanisms of early alcoholic liver injury will emerge which will lead to the development of new and more effective therapeutic strategies.